Effects of Various Factors on Carbonate Particle Growth Using Ureolytic Bacteria
2018 年 59 巻 9 号 p. 1520-1527
詳細
2018 年 59 巻 9 号 p. 1520-1527
In Microbially Induced Carbonate Precipitation (MICP), bacteria can perform metabolic activities that promote the deposition of carbonate particles in the form of calcite. Previously, purified urease and CaCl2 have been used for hydrolysis of urea to deposit carbonate particles. In our present study, Mg2+ ions were added to investigate the effect on the deposition of carbonate particles, because Mg2+ ions can delay the reaction rate and enhance the crystal deposition rate. Additionally, other parameters (temperature, solvent, bacterial population, and CaCl2 concentration) were taken into consideration to enhance the amount of carbonate deposition by ureolytic bacteria. The aim of this study was to investigate the mechanism of carbonate particle generation using urease producing bacteria (Pararhodobacter sp.) in laboratory test conditions using a translucent cell. In this study, marine ureolytic (Pararhodobacter sp.) bacteria were used and their urease activity was estimated considering bacterial concentration, temperature, and the effect of Ca2+ and Mg2+ ions. Digital microscopy analysis revealed the direct involvement of these parameters on the deposition of carbonate particles. The results of this study also showed that the type of deposited crystals, their shapes, and bacterial growth rate change depending on the medium used, the type of carbonate (metal ion used), CaCl2 concentration, and temperature. In addition, when Mg2+ and Ca2+ ions were used, the amount of particle deposition increased, which enhanced the possibility of becoming a superior binder for sand particles. This study is useful for the various sand solidification experiments and to regulate the most suitable conditions for engineering applications in future studies.
Microbially Induced Carbonate Precipitation (MICP) using urease-producing bacteria is a promising technique in the field of geotechnology and civil engineering. The mechanism of this technique is elucidated by the following reactions where urea is hydrolyzed by urease (ureolysis) to form ammonium ions and carbamate (eqs. (1)–(3)), which spontaneously hydrolyzes to form a second ammonium ion and bicarbonate. The reactions of urea hydrolysis and calcite formation are shown in eqs. (1)–(3).
| \begin{equation} \text{CO(NH$_{2}$)$_{2}$} + \text{2H$_{2}$O}\to \text{2NH$_{4}^{+}$} + \text{CO$_{3}^{2-}$} \end{equation} | (1) |
| \begin{equation} \text{CaCl$_{2}$}\to \text{Ca$^{2+}$} + \text{2Cl$^{-}$} \end{equation} | (2) |
| \begin{equation} \text{Ca$^{2+}$} + \text{CO$_{3}^{2-}$}\to \text{CaCO$_{3}$}\downarrow (\text{deposited}) \end{equation} | (3) |
Therefore, improving the cementation strength of deposited crystal particles’ structures is very important. Thus, this study aimed to determine the effect of various parameters (temperature, bacterial population, Ca2+ and Mg2+ ion concentration, and CaCl2 concentration) on crystal particle deposition to determine appropriate conditions for crystal particle deposition considering their shape, size, and diameter using digital microscopy image analysis, which can be effective for the practical applications from lab to engineering field scale.
In our study, Pararhodobacter sp. (identified by 16S rRNA gene analysis) isolated from the soil near beachrock in Okinawa, Japan were used for calcium carbonate deposition. ZoBell 2216 medium (5.0 g/L polypeptone, 1.0 g/L yeast extract, and 0.1 g/L FePO4 prepared in artificial seawater (Table 1), adjusted to pH 7.6–7.8) was used as the culture medium for cultivating the bacteria. The isolated bacteria were pre-cultured in 5 mL medium at 30°C with shaking at 160 rpm for 24 h. One millilitre of the pre-culture was inoculated into 100 mL of fresh ZoBell2216 medium incubated at 30°C with shaking at 160 rpm. During cultivation, cell concentration was determined by measuring the OD600 using a UV-vis spectrophotometer (V-730, JASCO Corporation, Tokyo, Japan) and deposited particle growth was investigated by digital microscopy (VHX-1000, Keyence Corporation, Tokyo, Japan).
Pararhodobacter sp. bacteria were cultivated in ZoBell2216E medium (100 mL of culture in a 300-mL Erlenmeyer flask) and cultivated for 3 days at 30°C in a column oven incubator. Then, an Erlenmeyer flask was filled with 1.0 M NaOH solution, pH adjusted to 7.6–7.8, and the top of the flask was covered with aluminum foil before autoclaving (KTS-2346 by ALP Corporation, Japan) at 121°C for 15 minutes and stored on a clean bench (CT-1200N-UV by Tanaka Seiki, Japan) for 24 hours to stabilize the precipitation (at room temperature: 25°C). Then, 1 mL of the prepared bacterial solution was transferred to a translucent cell (Fig. 1) designed for image analysis by a VHX-1000 digital microscope (Fig. 2). Ten deposited particles were randomly selected and the cell area was subjected to image analysis (0.5 cm × 0.5 cm). Image analysis was performed by manipulating the still image analysis software “Win-roof” to investigate the diameter (circle equivalent diameter) of deposited crystal grains and the area ratio (Fig. 3).
Translucent cell observation for carbonate precipitation.
Digital microscope VHX-1000.
Particle binarization for measuring diameter and inset picture showing diameter of a particle.
“Win-roof 2015” image analysis software was utilized to calculate the circle equivalent diameter of the deposited crystal particles. Figure 4 shows a schematic diagram of a graph of particle diameter over time. The vertical axis of the graph is the equivalent circle diameter and the horizontal axis is time. The deposition rate of crystal particles was calculated based on data in this graph. For the data analysis, the graphing software “Origin Pro 2015” was used. The average grain size (D) of deposited crystal grains was expressed by the following formula (4) and the growth rate of carbonate particles was calculated using the eqs. (4)–(8) which is illustrated in Fig. 4.
| \begin{equation} D = A\root 3\of{t} \end{equation} | (4) |
Schematic diagram of particle diameter over time (standard curve).
In this equation, the particle diameter (D) will continue to increase exponentially indefinitely over time. As a result, the growth reaction becomes steady and cannot advance. Therefore, according to chemical reaction formulas (1)–(3), it can be said that since the reaction of carbonate is considered to be almost the same as the decomposition rate of urea to be a constant rate, the following relationship expression can be obtained.
| \begin{equation} V = \alpha t \end{equation} | (5) |
(if the precipitated crystal particles are spherical in shape)
| \begin{equation} \text{Then},\ V = \frac{\pi}{6}D^{3} = \alpha t \end{equation} | (6) |
| \begin{equation} D^{3} = \frac{6\alpha}{\pi}t = A^{3}t \end{equation} | (7) |
| \begin{equation} \alpha = \frac{\pi}{6}A^{3}\ [\text{mm$^{3}$/h}] \end{equation} | (8) |
High concentrations of bacterial cells (from 106 to 108 cells) increased the amount of calcite deposition by MICP by enhancing the urease concentration for urea hydrolysis.16,17) Therefore, urea hydrolysis has a direct relationship with bacterial cell concentration for crystal particle deposition. Results of our study (Fig. 5) showed the deposition rate [%] of crystal particles to the number of bacteria (120 hours after the start of the test). The crystal deposition rate is the ratio of the total area of the particles on the photographed image. As shown in Figs. 5 and 12, crystal grain deposition was extremely affected by the number of bacteria and time. With increased time, crystal deposition and size also increased; the deposition of crystal grains was higher when the Bacteria/Solvent (distilled water) ratio was 1:9 compared to deposition when the Bacteria/Solvent (artificial sea water) ratio was 1:1 or Bacteria/Solvent (distilled water) ratio was 1:1. The increased bacterial concentration was measured by a UV-vis spectrophotometer at 600 nm and these results showed that the deposition rate of crystal particles was higher when the Bacteria/Solvent (both distilled water or artificial sea water) ratio was 1:9, compared to when the Bacteria/Solvent (both distilled water or artificial sea water) ratio was 1:1. Therefore, results from this experiment clearly showed that crystal deposition was greatly affected by the bacterial concentration and type of solvent. In this case, the largest deposited particle diameter was observed when the Bacteria/Solvent (distilled water) ratio was 1:9.
The deposition rate (α) of particles by the bacterial concentration.
Previous studies showed that calcium concentration can greatly accelerate CaCO3 deposition by microorganisms.5,12) It was revealed that CaCO3 deposition and CaCO3 precipitation consist of three distinct stages, are dependent on the calcium source, and that bacterial cells act as nucleation sites for crystal formation and growth.5) These results indicated that the calcium concentration influences crystal deposition. Therefore, it is necessary to investigate which calcium concentration is optimal for crystal deposition. Data shown in Figs. 6 and 7 shows that the crystal deposition rate is greatly affected by the concentration of bacteria and the CaCl2 concentrations of 0.1 M, 0.2 M, and 0.5 M. Moreover, the Bacteria/Solvent ratio also affects crystal deposition (Fig. 6). Data in Fig. 6 show that crystal deposition rate increased when bacteria and solvent concentration (artificial sea water) also increased. The crystal deposition rate also correlated with bacteria, solvent, and CaCl2 concentration (Fig. 7). Data in Fig. 7 show that crystal particles were uniform at concentrations of 0.1 M and 0.3 M and the average particle diameter was the largest at 0.5 M. This tendency was not seen at concentrations of 0.1 M and 0.3 M. Therefore, from this experiment, it can be stated that the number of crystal particles on the photographed image increased as the solvent, bacteria, and Ca2+ concentration increased, which are significant findings for the application of sand solidification.
The deposition rate (α) of precipitated crystals by CaCl2 concentration correspond with solvent and bacteria.
Effects of CaCl2 concentration on precipitated crystals correspond with solvent and bacteria.
Corresponding with the other enzymatic reactions, the catalysis of urea by urease depends on the temperature. The optimum temperature for activity of most ureases ranges from 20 to 37°C18,19) and the optimum range of the enzymatic reaction depends on environmental conditions and concentration of reactants in the system.20) In our study, data in Figs. 8 and 9 reveal the temperature dependence of the reaction. In each condition, three samples were placed at 25°C, 30°C, and 35°C, respectively. The average particle diameter at these temperatures is shown in Figs. 8 and 9. Figure 8 shows the deposition rate [%] of crystal particles at different temperatures after 48 h from the start of the test. The deposition rate is computed from the average particle diameter following eqs. (4)–(8). High urease activity was obtained for Pararhodobacter sp. at 20–60°C and a significant decrease in activity was observed above 70°C, ascribed to thermal denaturation of urease21) which is directly related to crystal deposition.5) The maximum urease activity of Pararhodobacter sp. was obtained at 60°C. In comparison with the previous studies, the number of deposited crystal particles on the photographed image was almost the same with little change depending on the bacterial concentration. As disclosed in Figs. 8 and 9, the crystal deposition rate was 27% at 25°C and 35°C, but in at 30°C, crystal deposition was considerably reduced by 19% and the average particle diameter was the largest at 25°C, followed by 35°C and 30°C with the lowest deposition rate (Fig. 9). At 30°C and 35°C, crystal growth stopped at about 12 h and reached a steady state, whereas growth at 25°C was stable for about 36 hours.
The deposition rate (α) of crystal particles by the temperature.
Effects of temperature on precipitated crystal particles correspond with solvent and bacteria.
It has been previously found that the maximum deposition ratio in the presence of Mg2+ was roughly 70% and the deposition ratio increased rapidly and approached the maximum, i.e., 90% when 10 and 20% of magnesium were used.22,23) Subsequently, Figs. 10 and 11 depict the influence of Mg2+ on crystal deposition. The testing conditions of “CaCl2+MgCl2” and CaCl2 were influenced by the basic solidification promoting solution (Table 1). Data in Figs. 10 and 11 shows that the average particle diameter was influenced by adding both Ca2+ and Mg2+. Furthermore, Fig. 10 shows the deposition rate [%] of crystal particles upon adding Mg2+ 48 h after the start of the test. The deposition rate was enumerated from the average particle diameter. Finally, the number of crystal particles on the photographed image was larger when MgCl2 was present in the solidification-promoting solution. In addition, as shown in Fig. 11, the average particle size was smaller and particle diameter growth stopped at about 12 h and become stationary, and the MgCl2 deposition rate of crystal particles was influenced by “Ca” and “Ca+Mg” solution, solvent, and bacterial concentration.
The deposition rate (α) of crystal particles by the “Ca” and “Ca+Mg” solidification solution.
Influence of “Ca” and “Ca+Mg” solidification solution on crystal deposition rate.
For CaCO3 deposition, bacterial concentration plays a vital role because the bacterial cells serve as nucleation sites for CaCO3 deposition and the availability of these nucleation sites is very significant for calcite depositions.24) Additionally, it was previously shown that 98% of the initial Ca2+ concentrations were deposited due to bacterial hydrolysis (microbially), but only 35% and 54% was deposited chemically in the water and subsequent medium, respectively.21,25) Therefore, for CaCO3 deposition and the diameter of the deposited crystal particles, bacterial concentration is very important because the bacterial cell concentration provides the nucleation sites for CaCO3 deposition and creates an alkaline environment for the induction of further calcite growth. Our experimental results indicate that the number and diameter of crystal particles increase with higher bacterial concentration, added promotion solution, and solvent, as shown in Fig. 5. The average particle diameter growth became steady earlier when bacterial concentration was lower compared to that when bacterial concentration was high. Finally, this result indicates how the deposited crystals are influenced by the bacterial concentration that is associated with the sand solidification test. In this experiment, the deposition rate was slightly lower, the number of deposited particles was larger, and the average particle diameter became smaller when bacterial concentration was high. This study also revealed that bacterial cell concentration greatly affects the bio-cementation of sand particles.
4.2 Effects of CaCl2 concentrationCaCl2 concentration is very important for CaCO3 deposition because it has been reported that the ideal calcium source and concentration affects CaCO3 deposition. However, high concentrations of CaCl2 (above 0.5 M) decreased the capability of calcite deposition and low concentrations (0.05–0.25 M) increased the capability of calcite deposition;20) this study also found that the optimum CaCl2 concentration for calcite deposition was 0.5 M and 0.25 M, respectively. Correspondingly, the amount of calcite deposition depends more on Ca2+ concentration than on urea concentration.5,11) As shown in Fig. 6, the crystal deposition rate was about 30% lower at a CaCl2 concentration of 0.5 M when distilled water was used as a solvent. In our study, the average particle diameter was smaller with artificial seawater and when Ca2+ concentration was high. However, a higher concentration of CaCl2 promoted the deposition rate diameter (Fig. 12) of deposited carbonate when distilled water was used as a solvent (Fig. 13). Additionally, when the concentration of “urea+CaCl2” was higher, the growth of microorganisms was inhibited. Therefore, from our study, it is seen that conditions with an extremely high CaCl2 concentration do not have potential for the deposition of crystal carbonates.
Crystal deposition at certain times of interval with bacterial concentration.
Deposited crystals diameter change with time.
In general, urease activity is increased by about 5 to 10 times when the temperature increased from 15°C to 20°C and 10°C to 20°C, respectively22,24) and the kinetic rate of urease hydrolysis26) for CaCO3 deposition was completely stable at 35°C, but when the temperature increased to 55°C, the enzymatic activity declined by almost 47%. Therefore, the temperature is a very significant parameter for urea hydrolysis and CaCO3 deposition, which is a major concern in this study because as the temperature is higher, the deposition rate of carbonate also increases.27) In this study, data in Fig. 8 show that the deposition rate was about 70% at 35°C where Bacteria/Artificial sea water ratio was 1:9. On the other hand, the deposition rate was similar between the artificial seawater and distilled water with changing temperatures, whereas the use of artificial seawater was observed to slow deposition rate at 25°C (Fig. 9). This is because the hydrolysis by microorganisms had decelerated due to the changes in cell growth at a low temperature of 25°C compared to growth at 35°C.
4.4 Effects of Mg2+It was previously reported that low concentrations of magnesium ion (i.e., 0.02 and 0.04 mol/L) significantly promoted the formation of aragonite and the substitution of a small amount of magnesium ion conveys a significant improvement in the deposition ratio.23) Furthermore, the acceleration of calcite deposition may be because the Mg2+ ions engaged in the reaction and the addition of magnesium also affects the shape of the deposited materials.14,27) In our experiment, we added MgCl2, and in agreement with the above results, the number of particles and deposition rate and diameter of crystals were decreased in the basic solidification-promoting solution containing MgCl2 (Fig. 10 and Fig. 11). In addition, the average particle diameter was larger when MgCl2 was present in the solidification-promoting solution. This is likely because the existence of multivalent cations suppresses its deposition rate and the crystal gradually grows, consequently, the crystal seems to be rounded. In addition, in this study, we found that the deposition speed was slower when Mg2+ was scarce in the solidification-promoting solution. Therefore, Mg2+ enhances the crystal deposition speed.
4.5 Summary of the discussionsIn brief, this study found that carbonate deposition and crystal formation are greatly affected by temperature and coexisting ions. Based on the findings obtained in this study, the results summarizing the degree of influence on carbonate particle deposition are shown in Table 3. For each item, a ◎ mark in the table represents a strong influence of the parameter, a ○ mark signifies the moderate influence, and a △ indicates a parameter with low influence. From the results of this study, we found that bacterial concentration, temperature, Mg–Ca ion, and solvent played a significant role in CaCO3 crystal deposition (spherical type) and the optimum conditions of these parameters (Table 3 and Table 4) can be applied for engineering applications in sand solidification tests towards soil improvement techniques.
Our results effectively demonstrate that the feasibility of using “Ca” and “Mg–Ca” ions was considerably influenced by bacterial concentration, temperature, and solvent for crystal carbonate deposition using marine bacteria species. In this work, Mg2+ ions were added and other parameters (temperature, solvent, bacterial population, and CaCl2 concentration) were taken into consideration to investigate the rate and the amount of carbonate deposition. The effects of Mg2+ on the deposited materials were analyzed to investigate the microstructure of the deposited materials by a digital microscope. Adding Mg2+ ions increased the deposition ratio of carbonate up to 80% with the CaCl2 concentration. The existence of “Ca” and “Mg–Ca” changed the shape and the size of the deposited crystal carbonate materials. This study can be useful for determining the suitable conditions for sand solidification by MICP method that can be applied to various ground improvement and engineering sectors.
This work was partially supported by JSPS KAKENHI, Grant number JP16H04404, Japan.
